Various patents and other publications are referenced throughout the specification. The disclosure of each of these publications is incorporated in its entirety by reference herein.
It is well known that the use of chemotherapy and radiotherapy to treat cancer patients is associated with severe side effects due to the toxicity of such treatments to cells, particularly epithelial cell populations, including stem cells, within the hair follicle, skin epidermis, and gastrointestinal mucosa.
Currently, there are no treatments to prevent cancer therapy side effects. Effective treatments would likely include molecules that i) inhibit or slow growth of the at-risk cells, ii) modify the cellular DNA of the at-risk cells to make it less easily damaged, and iii) provide some means with which to scavenge electrophilic drug metabolites or oxygen radicals formed during irradiation.
Polyamines and other amine compounds have been proposed as growth regulators. DENSPM, a synthetic analog of spermine, has been shown to decrease cell growth (Kramer et al., Cancer Res. 57:5521-5527, 1997), and has been studied in an early stage clinical trial as an antineoplastic drug (Creaven, P. et al., Invest. New Drugs 15:227-234, 1997; Streiff, R and Bender, J. Invest. New Drugs 19:29-39, 2001). The clinical trials, however, were aborted because of the serious side effects in multiple organ sites that were associated with the systemic use of this polyamine analog. These results teach that molecules used to decrease division of healthy stem cells that are at risk from cancer therapy would need to create a transient cell cycle block and would need to be applied topically to achieve local delivery to epithelial cells, with little or no systemic delivery, or if any, low enough to preclude protection of systemic cancer cells or induction of systemic side effects.
Naturally occurring polyamines, such as spermine, have been shown to bind to nucleic acids and to induce structural changes in helical DNA (Basu, H. and Marton, L., Biochem. J. 244:243-246, 1987; Feuerstein, B. et al., Nuc. Acids Res. 17:6883-6892, 1989). This binding has been suggested to occur through interaction of the positively-charged amine groups in the polyamine backbone and negatively-charged sites on the DNA backbone. Because of the manner in which electrophilic chemotherapy drugs or oxygen radicals generated by radiotherapy attack helical B-DNA within cells, the ability of polyamines to bind DNA and disrupt normal B-DNA structure could be helpful in protecting DNA within cells to which a polyamine was delivered.
An additional strategy for protecting cells against electrophiles/radicals has been to augment levels of the naturally occurring cellular nucleophile, glutathione (GSH). Both animal and cell culture studies have shown that there is a direct relationship between the intracellular concentration of GSH and the amount of exogenously administered alkylating molecule that is needed to achieve cell kill (Ho, D. and Fahl, W., J. Biol. Chem. 259:11231-11235, 1984; Ellouk-Achard, S. et al., Arch. Toxicol. Suppl. 17:209-214, 1995). Efforts to exogenously administer GSH to cells as a protectant have failed because mammalian cells are generally unable to take up this nucleophile. There have been efforts to modify the GSH molecule to enable cellular uptake, but these have not found clinical use.
Amifostine (WR-2721), a small molecule amine containing a thiophosphate group that is presumably converted to a thiol in cells, has been used systemically as a radio- and chemoprotectant with mixed results. Though it may provide free —SH groups within cells, it is not known to contain activity as either a growth regulator or as a modifier of DNA structure. The active metabolite of WR-2721, WR-1065, has been shown to be active as a radioprotector when added to cells in tissue culture, as has another small molecule, cysteamine (Purdie, J. W., Radiation Research 77:303-311, 1979). U.S. Pat. No. 6,239,119 (and published U.S. Patent Application No. 2003/0022867 A1) to Stogniew and Bourhis suggests the use of WR-2721, WR-1065 and their related metabolites as topical radio- or chemo-protectors. The published literature, however, contains little information about topical use of such small molecules, and the information that is published teaches the failure of these compounds as topically-applied protector molecules. Lowy et al. (Radiation Biology 105:425-428, 1972) reported that although systemic amifostine was effective in reducing radiation-induced skin damage in mice, topical application of the drug showed no protective effect. Verhey et al., (Radiation Research 93:175-183, 1983) also reported no protective effect when amifostine was applied topically to mouse skin.
Edwards et al. (U.S. Pat. Nos. 5,217,964 and 5,434,145) described the synthesis of short, spermidine- or spermine-like polyamine molecules that were modified to contain an alkyl-thiophosphate or alkyl-thiol group. In U.S. Pat. No. 5,217,964, the attached thiophosphate group (i.e., —SPO3H2) would require enzymatic activation by cellular phosphatases to form the nucleophilic —SH group. The alkyl-thiophosphate group(s) was bound to the polyamine molecule through a terminal benzyl ring and/or through one or more of the amines in the polyamine backbone. Polyamines containing aromatic rings have been described as structural inhibitors of the membrane polyamine transporter in mammalian cells. Such polyamines have been shown not to be transported into cells. In U.S. Pat. No. 5,434,145, Edwards showed bonding of alkyl-thiophosphate or alkyl-thiol groups to one or more of the backbone amines that are present in the short polyamine molecules. By modifying the secondary amines in the polyamine backbone with alkyl-thiophosphate groups, the amines were converted to tertiary amines, and this markedly altered the basicity of the individual modified amine, as well as that of the overall polyamine molecule. The attenuated basicity of the individual amine groups was accompanied by an alteration in three dimensional structure at these sites. With added alkyl functionality on the amine nitrogen atoms, steric bulkiness was increased, so the ability or freedom of the molecule to rotate and twist at these sites was markedly reduced. The altered basicity and steric constraints in these short spermine-like polyamines was surmised to perturb DNA binding by the modified polyamines, as compared to that of their natural polyamine counterparts. Consistent with this (DNA binding is a biological activity of natural polyamines), Edwards provided no information regarding biological activity for any of the structures proposed in U.S. Pat. No. 5,217,964 or 5,434,145. Similarly in U.S. Pat. No. 5,292,497, Schein et al. describe a method of reducing chemotherapy toxicity involving oral administration of S-3-(3-methylaminopropylamino) propyl dihydrogen phosphorothioate compounds.
Accordingly, there is a need in the art to create polyamine and other amine-based molecules that are optimized to achieve: i) local and transient growth regulation, ii) disruption of normal helical DNA structure upon binding, and iii) delivery and display of nucleophilic or other functional moieties within cells to enable scavenging of reactive electrophiles and radicals. There would be great advantage in developing compounds that can be used topically to prevent or diminish the toxic side effects of cancer chemotherapy and radiotherapy.